Modulation of voltage-dependent Ca2+ channels in rabbit colonic smooth muscle cells by c-Src and focal adhesion kinase.
There is emerging evidence indicating that smooth muscle contraction and Ca2+ influx through voltage-dependent L-type Ca2+ channels are regulated by tyrosine kinases; however, the specific kinases involved are largely unknown. In rabbit colonic muscularis mucosae cells, tyrosine-phosphorylated proteins of approximately 60 and 125 kDa were observed in immunoblots using an anti-phosphotyrosine antibody and were identified as c-Src and focal adhesion kinase (FAK) by immunoblotting with specific antibodies. FAK co-immunoprecipitated with c-Src, and the phosphorylation of the c-Src.FAK complex was markedly enhanced by platelet-derived growth factor (PDGF) BB. The presence of activated c-Src in unstimulated cells was identified in cell lysates by immunoblotting with an antibody recognizing the autophosphorylated site (P416Y). In whole-cell patch-clamp studies, intracellular dialysis of a Src substrate peptide and anti-c-Src and anti-FAK antibodies suppressed Ca2+ currents by 60, 62, and 43%, respectively. In contrast, intracellular dialysis of an anti-mouse IgG or anti-Kv1.5 antibody did not inhibit Ca2+ currents. Co-dialysis of anti-c-Src and anti-FAK antibodies inhibited Ca2+ currents (63%) equivalent to dialysis with the anti-c-Src antibody alone. PDGF-BB enhanced Ca2+ currents by 43%, which was abolished by the anti-c-Src and anti-FAK antibodies. Neither the MEK inhibitor PD 098059 nor an anti-Ras antibody inhibited basal Ca2+ currents or PDGF-stimulated Ca2+ currents. The alpha1C subunit of the L-type Ca2+ channel co-immunoprecipitated with anti-c-Src and anti-phosphotyrosine antibodies, indicating direct association of c-Src kinase with the Ca2+ channel. These data suggest that c-Src and FAK, but not the Ras/mitogen-activated protein kinase cascade, modulate basal Ca2+ channel activity and mediate the PDGF-induced enhancement of L-type Ca2+ currents in differentiated smooth muscle cells.
Hu
X Q
XQ
Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Singh
N
N
Mukhopadhyay
D
D
Akbarali
H I
HI
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002469:Cell Separation; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D003956:Dialysis; D004553:Electric Conductivity; D051416:Focal Adhesion Protein-Tyrosine Kinases; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D017479:Receptors, Platelet-Derived Growth Factor / Q000378:metabolism; D015398:Signal Transduction; D018631:ras Proteins / Q000378:metabolism
D000906:Antibodies; D015220:Calcium Channels; D015815:Cell Adhesion Molecules; D010982:Platelet-Derived Growth Factor; D011505:Protein-Tyrosine Kinases; D017479:Receptors, Platelet-Derived Growth Factor; D051416:Focal Adhesion Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018631:ras Proteins; D002118:Calcium